Journal: PLoS ONE

Article Title: Generation of cryopreserved macrophages from normal and genetically engineered human pluripotent stem cells for disease modelling

doi: 10.1371/journal.pone.0250107

Figure Lengend Snippet: (A) A schematic representation of the differentiation process from iPSC to HPC with the timeline and cytokines utilized throughout the process. (B) Representative flow cytometry dot plots for the HPCs’ (b1) lymphoid, (b2) erythroid, and (b3) myeloid potential from 01279 iPSCs. (C) Quantification of HPC purity was confirmed by detection of, CD34, CD43, CD41, CD235a, and CD45 expression on HPCs derived from 01279, SNCA A53T, GRN R493X, and MECP2 HM is depicted. (D) Megakaryocytes/proplatelets stained using MegaCult-C kit. HPCs were plated in collagen-based assay (MegaCult) and to generate megakaryocytes capable of shedding pro-platelets. The emerging megakaryocytes were stained for the presence of CD41 or a matched isotype control and the staining was captured by alkaline phosphatase staining. Presence of megakaryocytes/proplatelets was confirmed by detection of glycoprotein IIb/IIIa accompanied by visualization of pink color. Representative isotype and megakaryocytes/proplatelets images captured using 20X magnification, (d1) 01279, (d2) SNCA A53T, (d3) GRN R493X, and (d4) MECP2 HM. (E) (e1) Multipotency of HPC quantified using the serum free methocult Colony Forming Unit (CFU) assay. The presence of erythroid (CFU-E/BFU-E), myeloid (CFU-M), granulocyte (CFU-G), granulocyte-macrophage (CFU-GM), and multipotent granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) were scored manually and the graphs represent average ± SE for each line. The graph depicts CFU from 01279 (n = 32± SE), SNCA A53T (n = 6± SE), GRN R493X (n = 6± SE), and MECP2 HM (n = 6± SE). A representation of emerging CFU colonies, CFU-E (e2), BFU-E (e3), CFU-G (e4), CFU-M (e5), CFU-GM (e6), and GEMM (e7).

Article Snippet: Purification of HPCs was achieved using Indirect CD34 MicroBead Kit (Miltenyi Systems).

Techniques: Flow Cytometry, Expressing, Derivative Assay, Staining, Collagen Assay, Control, Colony-forming Unit Assay

Journal: PLoS ONE

Article Title: Generation of cryopreserved macrophages from normal and genetically engineered human pluripotent stem cells for disease modelling

doi: 10.1371/journal.pone.0250107

Figure Lengend Snippet: (A) Schematic representation of the differentiation process from HPC to common myeloid progenitor (CMP) to macrophages under defined conditions. (B) Loss of CD34 expression and augmentation of HPC and CMP stage of differentiation for (b1) 01279, (b2) SNCA A53T, (b3) GRN R493X, and (b4) MECP2 HM KO. (C) Efficiency of generating macrophage from iPSCs lines. The efficiency calculated by total viable cell number of iPSC seeded and total number viable macrophages obtained at the end of the process.

Article Snippet: Purification of HPCs was achieved using Indirect CD34 MicroBead Kit (Miltenyi Systems).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Exploration of T cell immune responses by expression of a dominant-negative SHP1 and SHP2

doi: 10.3389/fimmu.2023.1119350

Figure Lengend Snippet: dSHP2 but not dSHP1 alleviates PD1-mediated suppression of CAR T activity. (A) Schematic of CAR constructs. Retroviral constructs were designed consisting of an anti-GD2 CAR with a CD28-CD3ζ endodomain linked to RQR8 as a marker of transduction and including PD1 and/or dSHP1 or dSHP2 separated by viral 2A sequences (shown in red). (B) Effect of dSHP1 (left panel) and dSHP2 (right panel) on PD1/PDL1-mediated suppression of CAR cytotoxicity. CAR T cells were co-cultured with 2x10 5 SupT1 cells lacking antigen expression (SupT1-NT) or engineered to express GD2 alone (SupT1-GD2) or in the presence of PDL1 (SupT1-GD2-PDL1) at an effector:target (E:T) ratio of 1:4 for 72 hours. Surviving target cells were enumerated and normalized to the respective co-cultures with non-transduced T cells (100%). (C, D) Effect of dSHP1 (left panel) and dSHP2 (right panel) on PD1/PDL1-mediated suppression of IFNγ and IL2 secretion. Supernatants from (B) were harvested and cytokine secretion measured by ELISA. (E) Effect of dSHP1 (left panel) and dSHP2 (right panel) on PD1/PDL1-mediated suppression of proliferation. CAR T cells were labelled with Cell Trace Violet and incubated with 2x10 5 of the indicated SupT1 target cells at an E:T ratio of 1:2 for 4 days and CAR T cells enumerated. Bars represent the mean of 5-6 biologically independent replicates. Statistical significance was measured by two-way ANOVA. Significance is defined as: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Antibodies used were: CCR7-PE (Miltenyi Biotec; 130-119-583), CD19–APC Cy7 (BioLegend; 302218), CD25-BV421 (Biolegend; 356114), CD27-VioBright515 (Miltenyi Biotec; 130-120-028), CD3–PE Cy7 (BioLegend; 344816), CD3-VioGreen (Miltenyi Biotec; 130-113-142), CD45RA-APCVio770 (Miltenyi Biotec; 130-117-747), CD69-FITC (Biolegend; 310904), CD8-APC-Cy7 (Biolegend; 301016), CD8-Vioblue (Miltenyi Biotec; 130-110-683), CD95-PEVio770 (Miltenyi Biotec 130-113-006), HA–AF488 (Biolegend; 901509), KLRG1-APC-Vio77 (Miltenyi Biotec; 130-120-423), LAG3-VioBright515 (Miltenyi Biotec; 130-120-012), PD1-PE (Miltenyi Biotec; 130-120-382), CD34 (RQR8)-PE (R&D Systems; FAB7227P), CD34 (RQR8)-APC (R&D Systems; FAB7227A), SYTOXTM AADvancedTM Dead Cell Stain (ThermoFisher; S10274), TIM3-PEVio770 (Miltenyi Biotec; 130-121-334), GD2-APC (Biolegend; 357306), HVEM-PE (Biolegend; 318806), PDL1-PE (Biolegend; 329706), CD48-PE (Biolegend; 336708), CD80-FITC (Biolegend; 305206), CD155-PE (Biolegend; 337610).

Techniques: Activity Assay, Construct, Retroviral, Marker, Transduction, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Stroke

Article Title: Trametinib decreased intracerebral hemorrhages and endothelial-to-mesenchymal transition in KRAS G12V -induced brain arteriovenous malformations in mice

doi: 10.1161/STROKEAHA.125.052418

Figure Lengend Snippet: (A) Immunostaining images of mesenchymal markers (N-cad, Slug, and CD44), endothelial marker (VE-Cad), (B) proliferation (Ki-67), and angiogenesis markers (CD34 and pVEGFR2), and the respective intensity in CD31+ intact and bAVM vessels. n=ROIs (dots) in 2–3 (Veh) or 3–6 (TM) mice (1mg/kg and 2mg/kg combined), Mice were sacrificed at 10 weeks after treatments, Data expressed as mean value of dots ± SEM, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 vs KRAS G12V mice treated with vehicle. Student’s t -test. Scale bar = 100μm. Veh, Vehicle; TM, Trametinib.

Article Snippet: The fixed samples were incubated with primary antibodies: CD31 (AF3628, R&D Systems, Minneapolis, MN), Ter119 (MAB1125, R&D systems), VE-Cad (361900, Thermo Fisher Scientific, Waltham, MA), p-ERK1/2 (9101S), N-Cad (13116S), Slug (9585S), CD44 (37259S), and Ki-67 (9129S) from Cell Signaling Technology, CD34 (NB6001071, Novus Biologicals, Centennial, CO), or pVEGFR2 (SAB4504567, Sigma-Aldrich, St. Louis, MO), followed by secondary antibodies: 488-conjugated donkey anti-goat (705-545-147) or anti-rabbit IgG (711-545-152), Rhodamine Red-X-AffiniPure donkey anti-mouse (715-295-151), anti-rabbit (711-295-152), or anti-goat IgG (705-295-147), 647-conjugated donkey anti-rabbit (711-605-152), anti-mouse (715-605-150), or anti-rat IgG (712-605-153).

Techniques: Immunostaining, Marker

Journal: Cells

Article Title: IMG-A1: A Novel Immortalized Granulosa Cell Line for Investigating FSH-Dependent Folliculogenesis and Ovarian Pathophysiology

doi: 10.3390/cells14241940

Figure Lengend Snippet: Basic Characterization of the IMG-A1 Granulosa Cell Line. ( a , b ) Phase-contrast micrographs of the IMG-A1 cell culture. ( c ) Staining for senescence-associated β-galactosidase activity in passage 18 cells. ( d – f ) Immunocytochemical staining for the proliferation marker Ki67, showing a phase-contrast image ( d ), Ki67 immunofluorescence ( e ), and a merged image with DAPI nuclear counterstain ( f ). ( g , h ) Representative metaphase spreads showing a normal diploid karyotype (40 chromosomes) ( g ) and a near-tetraploid karyotype (78 chromosomes) from the IMG-A1 line ( h ). ( i – k ) Ploidy analysis by flow cytometry. Control blood cells ( i ) and primary granulosa cells ( j ) show characteristic diploid (2n) and tetraploid (4n, G2/M phase) peaks. The IMG-A1 culture ( k ) displays a predominantly near-tetraploid and near-octaploid cell population. ( l ) Flow cytometry analysis confirming the homogeneity of the IMG-A1 culture (passages 10 and 40) based on staining for the stromal marker CD29 and the absence of hematopoietic markers CD45, CD34, and the fibroblast marker CD90. Quadrant gates were set based on unstained controls. Scale bars = 100 µm.

Article Snippet: PE CD34 Rat mAb [RAM34] , Elabscience Biotechnology Co., Ltd., Wuhan, China , E-AB-F1284D.

Techniques: Cell Culture, Staining, Activity Assay, Marker, Immunofluorescence, Flow Cytometry, Control